Clinical outcomes for sexual and gender minority adolescents in a dialectical behaviour therapy programme.

BACKGROUND: Sexuality and gender minoritised (SGM) adolescents are at increased risk of self-injury and suicide, and experience barriers to accessing mental health support. Dialectical behaviour therapy (DBT) is an effective treatment for self-injury and emotion dysregulation in adolescent populations, but few studies have published outcomes of DBT for SGM young people. AIMS: This study aimed to investigate treatment outcomes and completion for SGM adolescents and their cisgender and heterosexual peers, in the National & Specialist CAMHS, DBT service (UK). METHOD: Treatment completion, and opting out before and during treatment were examined for sexual and gender identity groups, as well as changes by the end of treatment in emotion dysregulation, self-injury, in-patient bed-days, emergency department attendances, and borderline personality disorder, depression and anxiety symptoms. RESULTS: SGM adolescents were over-represented in this service, even after considering their increased risk for self-injury. No statistically significant differences were found for treatment completion between the sexual orientation and gender identity groups, although there were patterns indicating possible lower treatment uptake and completion that warrant further investigation. Clinical outcomes for treatment-completers showed improvement by the end of DBT for each group, with few exceptions. DISCUSSION: These results are from relatively small subsamples, and it was not possible to separate by sex assigned at birth. Findings should be treated tentatively and as early indications of effect sizes to inform future studies. This study suggests that DBT could be a useful treatment for SGM adolescents in a highly specialist treatment setting.

Gender- and Sexuality-Minoritised Adolescents in DBT: A Reflexive Thematic Analysis of Minority-Specific Treatment Targets and Experience.

Gender- and sexuality-minoritised (GSM) adolescents are at increased risk of self-harm and suicidal behaviours compared to their cisgender and heterosexual peers. This increased risk is thought to be explained in part by exposure to stigma and societal oppression. Dialectical Behaviour Therapy (DBT) is an evidence-based intervention for self-harm and suicidal behaviour that may have advantages for supporting GSM young people in distress. No study has yet sought to understand what GSM-associated difficulties may be important to consider in DBT for adolescents, or the experiences of GSM young people in a standard DBT programme. Therefore, this study aimed to understand the experiences of GSM young people in DBT and what difficulties and dilemmas associated with their gender and sexuality diversity were thought by them to be important to target in DBT. Qualitative interviews were conducted with 14 GSM young people in a comprehensive DBT programme and were analysed using Reflexive Thematic Analysis. The analysis was supported by two further GSM young people who had finished DBT. The findings were split into three overarching themes (Identity, Impact of Others, and Space for Sexual and Gender Identity in DBT), each with themes within. The identity-based theme included "identity confusion and acceptance"; the relationship-based themes included "cis-Heterosexism" and "community connectedness"; and the space within DBT themes included "negotiating focus and targeting in DBT" and "creating safety in DBT". Findings are discussed in relation to implications and recommendations for therapists working with GSM young people within and outside of DBT.

What is a cell type?

A next step for cell atlases should be to chart perturbations in human model systems.

Multimodal spatiotemporal phenotyping of human retinal organoid development.

Organoids generated from human pluripotent stem cells provide experimental systems to study development and disease, but quantitative measurements across different spatial scales and molecular modalities are lacking. In this study, we generated multiplexed protein maps over a retinal organoid time course and primary adult human retinal tissue. We developed a toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components and global patterning in each organoid and primary tissue. In addition, we generated a single-cell transcriptome and chromatin accessibility timecourse dataset and inferred a gene regulatory network underlying organoid development. We integrated genomic data with spatially segmented nuclei into a multimodal atlas to explore organoid patterning and retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death and showing that mosaic genetic perturbations in retinal organoids provide insight into cell fate regulation.

Human-specific genetics: new tools to explore the molecular and cellular basis of human evolution.

Our ancestors acquired morphological, cognitive and metabolic modifications that enabled humans to colonize diverse habitats, develop extraordinary technologies and reshape the biosphere. Understanding the genetic, developmental and molecular bases for these changes will provide insights into how we became human. Connecting human-specific genetic changes to species differences has been challenging owing to an abundance of low-effect size genetic changes, limited descriptions of phenotypic differences across development at the level of cell types and lack of experimental models. Emerging approaches for single-cell sequencing, genetic manipulation and stem cell culture now support descriptive and functional studies in defined cell types with a human or ape genetic background. In this Review, we describe how the sequencing of genomes from modern and archaic hominins, great apes and other primates is revealing human-specific genetic changes and how new molecular and cellular approaches - including cell atlases and organoids - are enabling exploration of the candidate causal factors that underlie human-specific traits.

EPIREGULIN creates a developmental niche for spatially organized human intestinal enteroids.

Epithelial organoids derived from intestinal tissue, called enteroids, recapitulate many aspects of the organ in vitro and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identified an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells and feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown enteroids, and EREG-grown enteroids showed that EGF enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.

Inferring and perturbing cell fate regulomes in human brain organoids.

Self-organizing neural organoids grown from pluripotent stem cells1-3 combined with single-cell genomic technologies provide opportunities to examine gene regulatory networks underlying human brain development. Here we acquire single-cell transcriptome and accessible chromatin data over a dense time course in human organoids covering neuroepithelial formation, patterning, brain regionalization and neurogenesis, and identify temporally dynamic and brain-region-specific regulatory regions. We developed Pando-a flexible framework that incorporates multi-omic data and predictions of transcription-factor-binding sites to infer a global gene regulatory network describing organoid development. We use pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoids. We find that certain factors regulate the abundance of cell fates, whereas other factors affect neuronal cell states after differentiation. We show that the transcription factor GLI3 is required for cortical fate establishment in humans, recapitulating previous research performed in mammalian model systems. We measure transcriptome and chromatin accessibility in normal or GLI3-perturbed cells and identify two distinct GLI3 regulomes that are central to telencephalic fate decisions: one regulating dorsoventral patterning with HES4/5 as direct GLI3 targets, and one controlling ganglionic eminence diversification later in development. Together, we provide a framework for how human model systems and single-cell technologies can be leveraged to reconstruct human developmental biology.

A nomenclature consensus for nervous system organoids and assembloids.

Self-organizing three-dimensional cellular models derived from human pluripotent stem cells or primary tissue have great potential to provide insights into how the human nervous system develops, what makes it unique and how disorders of the nervous system arise, progress and could be treated. Here, to facilitate progress and improve communication with the scientific community and the public, we clarify and provide a basic framework for the nomenclature of human multicellular models of nervous system development and disease, including organoids, assembloids and transplants.

Single-cell analyses of axolotl telencephalon organization, neurogenesis, and regeneration.

Salamanders are tetrapod models to study brain organization and regeneration; however, the identity and evolutionary conservation of brain cell types are largely unknown. We delineated the cell populations in the axolotl telencephalon during homeostasis and regeneration using single-cell genomic profiling. We identified glutamatergic neurons with similarities to amniote neurons of hippocampus, dorsal and lateral cortex, and conserved γ-aminobutyric acid-releasing (GABAergic) neuron classes. We inferred transcriptional dynamics and gene regulatory relationships of postembryonic, region-specific neurogenesis and unraveled conserved differentiation signatures. After brain injury, ependymoglia activate an injury-specific state before reestablishing lost neuron populations and axonal connections. Together, our analyses yield insights into the organization, evolution, and regeneration of a tetrapod nervous system.

Characterization of RNA content in individual phase-separated coacervate microdroplets.

Condensates formed by complex coacervation are hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of biological condensates and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and to which extend single condensates differ in their RNA composition. To address this, we developed an approach to read the RNA content from single synthetic and protein-based condensates using high-throughput sequencing. We find that certain RNAs efficiently accumulate in condensates. These RNAs are strongly enriched in sequence motifs which show high sequence similarity to short interspersed elements (SINEs). We observe similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

High temporal resolution proteome and phosphoproteome profiling of stem cell-derived hepatocyte development.

Primary human hepatocytes are widely used to evaluate liver toxicity of drugs, but they are scarce and demanding to culture. Stem cell-derived hepatocytes are increasingly discussed as alternatives. To obtain a better appreciation of the molecular processes during the differentiation of induced pluripotent stem cells into hepatocytes, we employ a quantitative proteomic approach to follow the expression of 9,000 proteins, 12,000 phosphorylation sites, and 800 acetylation sites over time. The analysis reveals stage-specific markers, a major molecular switch between hepatic endoderm versus immature hepatocyte-like cells impacting, e.g., metabolism, the cell cycle, kinase activity, and the expression of drug transporters. Comparing the proteomes of two- (2D) and three-dimensional (3D)-derived hepatocytes with fetal and adult liver indicates a fetal-like status of the in vitro models and lower expression of important ADME/Tox proteins. The collective data enable constructing a molecular roadmap of hepatocyte development that serves as a valuable resource for future research.

Spatial transcriptomic and single-nucleus analysis reveals heterogeneity in a gigantic single-celled syncytium.

In multicellular organisms, the specification, coordination, and compartmentalization of cell types enable the formation of complex body plans. However, some eukaryotic protists such as slime molds generate diverse and complex structures while remaining in a multinucleate syncytial state. It is unknown if different regions of these giant syncytial cells have distinct transcriptional responses to environmental encounters and if nuclei within the cell diversify into heterogeneous states. Here, we performed spatial transcriptome analysis of the slime mold Physarum polycephalum in the plasmodium state under different environmental conditions and used single-nucleus RNA-sequencing to dissect gene expression heterogeneity among nuclei. Our data identifies transcriptome regionality in the organism that associates with proliferation, syncytial substructures, and localized environmental conditions. Further, we find that nuclei are heterogenous in their transcriptional profile and may process local signals within the plasmodium to coordinate cell growth, metabolism, and reproduction. To understand how nuclei variation within the syncytium compares to heterogeneity in single-nucleus cells, we analyzed states in single Physarum amoebal cells. We observed amoebal cell states at different stages of mitosis and meiosis, and identified cytokinetic features that are specific to nuclei divisions within the syncytium. Notably, we do not find evidence for predefined transcriptomic states in the amoebae that are observed in the syncytium. Our data shows that a single-celled slime mold can control its gene expression in a region-specific manner while lacking cellular compartmentalization and suggests that nuclei are mobile processors facilitating local specialized functions. More broadly, slime molds offer the extraordinary opportunity to explore how organisms can evolve regulatory mechanisms to divide labor, specialize, balance competition with cooperation, and perform other foundational principles that govern the logic of life.

Lineage recording in human cerebral organoids.

Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enable highly resolved descriptions of cell states within these systems; however, approaches are needed to directly measure lineage relationships. Here we establish iTracer, a lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. We apply iTracer to explore clonality and lineage dynamics during cerebral organoid development and identify a time window of fate restriction as well as variation in neurogenic dynamics between progenitor neuron families. We also establish long-term four-dimensional light-sheet microscopy for spatial lineage recording in cerebral organoids and confirm regional clonality in the developing neuroepithelium. We incorporate gene perturbation (iTracer-perturb) and assess the effect of mosaic TSC2 mutations on cerebral organoid development. Our data shed light on how lineages and fates are established during cerebral organoid formation. More broadly, our techniques can be adapted in any iPSC-derived culture system to dissect lineage alterations during normal or perturbed development.

A roadmap for the Human Developmental Cell Atlas.

The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.

NGN2 induces diverse neuron types from human pluripotency.

Human neurons engineered from induced pluripotent stem cells (iPSCs) through neurogenin 2 (NGN2) overexpression are widely used to study neuronal differentiation mechanisms and to model neurological diseases. However, the differentiation paths and heterogeneity of emerged neurons have not been fully explored. Here, we used single-cell transcriptomics to dissect the cell states that emerge during NGN2 overexpression across a time course from pluripotency to neuron functional maturation. We find a substantial molecular heterogeneity in the neuron types generated, with at least two populations that express genes associated with neurons of the peripheral nervous system. Neuron heterogeneity is observed across multiple iPSC clones and lines from different individuals. We find that neuron fate acquisition is sensitive to NGN2 expression level and the duration of NGN2-forced expression. Our data reveal that NGN2 dosage can regulate neuron fate acquisition, and that NGN2-iN heterogeneity can confound results that are sensitive to neuron type.

Charting human development using a multi-endodermal organ atlas and organoid models.

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.

Resolving organoid brain region identities by mapping single-cell genomic data to reference atlases.

Self-organizing tissues resembling brain structures generated from human stem cells offer exciting possibilities to study human brain development, disease, and evolution. These 3D models are complex and can contain cells at various stages of differentiation from different brain regions. Single-cell genomic methods provide powerful approaches to explore cell composition, differentiation trajectories, and genetic perturbations in brain organoid systems. However, it remains a major challenge to understand the heterogeneity observed within and between individual organoids. Here, we develop a set of computational tools (VoxHunt) to assess brain organoid patterning, developmental state, and cell identity through comparisons to spatial and single-cell transcriptome reference datasets. We use VoxHunt to characterize and visualize cell compositions using single-cell and bulk genomic data from multiple organoid protocols modeling different brain structures. VoxHunt will be useful to assess organoid engineering protocols and to annotate cell fates that emerge in organoids during genetic and environmental perturbation experiments.